Binding assays are in wide use in laboratories for the detection and measurement of analytes in samples. For biological samples such as urine, whole blood, plasma, serum, and other biological fluids, assays are often performed in hospitals and clinical laboratories. Binding assays can also be performed in environmental, agricultural, veterinary, industrial, athletic, and legal/forensic settings. The principles involved in such assays are well known by those skilled in the art. Many such devices have been described and are available commercially.
For convenience and to facilitate description and understanding of the novel devices of this invention, it will principally be described as applied to immunological assays in which the analyte is an antigen, the detection reagent is a labeled antibody, and the capture molecule is another antibody. The skilled artisan will recognize that the principles and practices described can be usefully employed to analyze a wide variety of analytes, including:
Oncology antigens. Certain antigenic compounds from human subjects are found in abnormal quantities or abnormal locations in the body. For example, the presence of elevated levels of antigens such as prostate specific antigen (PSA) and carcinoembryonic antigen (CEA) can be early indicators of tumorigenic processes.
Other antigens. Certain other naturally occurring antigens in the body can be indicative of disease processes. For example, the presence of human hemoglobin in human fecal occult blood samples can be indicative of gastrointestinal disorders.
Hormones. Human chorionic gonadotropin (hCG) is frequently assayed as a test for pregnancy.
Antigenic determinants of infectious disease organisms, including bacteria, fungi, viruses, and yeast. Common assays include assays for antigens known to be associated with infection by hepatitis, HIV, human papillomavirus (HPV), malaria, West Nile Virus, Mycobacterium tuberculosis, and Helicobacter pylori. 
Antibodies raised by the immune system against antigens found on infectious disease organisms. Common assays include assays for antibodies against the hepatitis group of viruses, HPV, HIV, Helicobacter pylori, and malaria.
Drugs and metabolites thereof. Assays have been used to monitor the therapeutic efficacy of the drug. Assays can also be used to assess illegal usage of legal drugs, for example in the athletic context, as well as usage of illegal drugs.
Vitamins and metabolites thereof.
Enzymes. The absence or diminished quantities of certain enzymes can be diagnostically important in the inherited metabolic diseases, for example histidase as a marker of histidinemia, and hexosaminidase as a marker of Tay-Sachs disease.
Tissue specific antigens. The presence or elevated level of a tissue specific antigen in circulation may often be an indication of tissue damage. For example, cardiac troponin is a marker of myocardial infarction, and creatine kinase is a marker of muscle damage.
There are a few common types of immunological binding assay formats, such as described in Ching et al, U.S. Pat. No. 5,120,643, the contents of which are incorporated herein by reference. The first are competitive binding assays, which can be used to determine the concentration of an analyte in a sample solution. In competitive binding assays, known quantities of labeled reagents and unlabeled analytes for example labeled and unlabeled antigens or antibodies compete for binding sites on an immobilized binding material. After an incubation period, unbound materials are washed away from the system, and a measurement is made of the amount of labeled reagent bound to the binding material. The measurement is made by comparing the amount of binding to one or more known reference standards. In this way, the concentration of the analyte in the sample may be determined.
A second type of immunological binding assay is the sandwich assay. This assay format typically involves a porous media having a mobilizable labeled antibody and an immobilized unlabeled antibody partner for the analyte of interest in the biological sample. These antibodies are often referred to as the conjugate or detection antibody and the capture antibody. The sample is added to the porous media, to allow for formation of labeled mobilizable product which moves along the porous media to contact and react with the capture antibody to form a fixed, detectable, concentrated capture antibody/analyte/detection reagent complex. Sandwich assays may include immunological assays wherein the labeled reagent and the second binding partner are both antibodies, or are both antigens, and may also include other types of molecules. For example, enzyme immunoassays (EIA) and enzyme-linked immunosorbent assays (ELISA) are types of sandwich immunoassays, in which the binding is between an antibody and an antigen, and the labeling partner is an enzyme.
Many of these binding assays can be conducted in liquid phase or in modified liquid phase by applying liquids to a solid substrate—i.e. test tubes, microtiter plates or other similar bulk formats in which reagents are added sequentially and directly by a laboratorian. However, in clinical laboratories the use of solid phase chromatographic binding assay devices has become commonplace for their relative ease of use, economy, and reproducibility.
Typically, these chromatographic assay devices are comprised of a porous chromatographic medium which acts as the matrix for the binding assay. The sample of interest is added directly or indirectly to one end of the medium, and is chromatographically transported to a detection reagent with which it reacts to form a labeled product, which is then transported to a test zone containing an immobilized capture reagent such as a capture antibody, in which the presence, absence, or quantity of an analyte of interest can be determined.
For example, Deutsch et al. (U.S. Pat. Nos. 4,094,647; 4,235,601; and 4,361,537), the contents of which are incorporated herein by reference, describe binding assays where detection and capture reagents are deposited on a test strip with appropriate spacing in between them. Upon application of the sample, the reagents react and the product is transported to the test zones by chromatographic solvent transport. The Deutsch et al. devices include a retarding agent which slows transport of either the analyte of interest or of a product including the analyte of interest.
Ching et al. (U.S. Pat. Nos. 5,120,643 and 6,534,320), the contents of which are incorporated herein by reference, describe a test strip assay device in which a mobile conjugate labeled with colloidal labels such as gold, can be deposited on a chromatographic medium, and after reaction with an analyte, thus transported with the solvent to a test zone. Ching et al. describe a zone containing a dried conjugate. The conjugate/reagent product is solubilized by the movement of the sample down the strip toward the test zone. In some embodiments of the Ching et al device, the labeled reagent can be deposited on the test strip downstream of the test zone, and in others it can be premixed with the sample prior to its application onto the strip.
Ching et al. also teach the use of chromatographic transport “facilitating agents” which are stated to promote chromatographic transport, and to prevent aggregation and inactivation of specific binding materials and reagents in solution. The facilitating agents include polyethylene glycol, meta-soluble proteins such as casein, and detergents such as SDS.
May et al. (U.S. Pat. Nos. 5,622,871; 5,656,503; 6,187,598; and 6,228,660), the contents of which are incorporated herein by reference, describe a home pregnancy test in which the labeled mobilizable detection reagent reacts with an analyte and the resulting product migrates with the liquid sample as the sample progresses to the test zone. During manufacture, after the unlabeled binding agent (an anti-hCG antibody) is added to and immobilized in the test zone, the remainder of the test strip material is treated with blocking agents, in order to block any remaining binding sites. Suitable blocking agents are described as protein and polyvinylalcohol. Additional manufacturing steps include the addition of a glaze of aqueous sugar or cellulose solution onto the test strip in the region where the labeled capture reagent will be deposited. May et al. hypothesize that the glazing prevents interaction between the detection reagent and the test strip. After the glazing step, the detection reagent is deposited on the test strip.
Chandler et al. (U.S. Pat. Nos. 6,168,956; 6,017,767; 5,998,220; 5,877,028; 5,869,345; 5,846,838; 5,648,274; 5,607,863; 5,468,648; and 6,528,321), the contents of which are incorporated herein by reference, describes an assay device with opposable elements in a flexible or hinged book structure. For example, U.S. Pat. No. 5,846,838 describes a device in which a first opposable element contains a sample preparation zone, and in which the second opposable element contains the chromatographic medium. Chandler et al. describe bringing the two opposable portions of the test into contact with each other functions to apply the sample to the chromatographic medium, thereby starting the test. Chandler et al. use the first opposable element to bring certain reagents into contact with the chromatographic medium. The Chandler devices describe movement of the sample through the test zone of the chromatographic medium at the same time as the labeled reagent.
Fitzgerald et al. (U.S. Pat. No. 6,528,321), the contents of which are incorporated herein by reference, describe a chromatographic device having two opposable elements. The first element contains a sample application zone which consists of a porous matrix material capable of trapping the cellular components of blood; and a chromatographic medium comprising a test zone with a specific binding partner for the analyte of interest and further comprising a detection reagent zone with a labeled second specific binding partner for the analyte, which can be resolubilized by the solvent front passing through the porous material. The second opposable element contains an applicator and an absorber. When the two opposable elements are brought into contact with each other by the operator, the applicator releases a wash solution onto the first opposable element. The Fitzgerald et al. device, like the Ching et al. device, provides that the labeled reagent flow through at least a portion of the chromatographic medium contemporaneously with the sample.
A careful analysis of the patents listed above reveals that except for the Chandler devices, a principal feature is that they employ lateral flow of the samples which are usually in the same planes through a detection zone and a capture zone which are in substantially the same plane. Another feature is that the analyte in the sample first migrates to a detection zone where the analyte reacts with a labeled detection reagent and the resulting product then migrates to the test zone to form and concentrate a detectable complex. The hinged devices are somewhat different from the standard structures in that the reactants are not in a lateral line. They are initially in parallel planes in the two components. The components are brought into opposition to permit the detection reagent/ analyte product to react with a capture reagent to form the detectable complex. In all structures the first reaction is between the analyte and the detection reagent in approximately the same or parallel planes and the resulting detection reagent/analyte product thereafter contacts and reacts with the capture reagent to form a detectable complex.
Currently available chromatographic assay devices such as described in the above patents suffer from some drawbacks and limitations. Most commercially available porous membranes have some innate capacity for non-specific binding. Many of the prior art assay devices utilize extrinsic blocking agents to minimize non-specific binding of the labeled reagent to the test strip. Non-specific binding between labeled reagent and the test strip can lower the sensitivity and reproducibility of the test, and can also create an undesirable background of labeled reagent which renders reading a color reaction more difficult. Blocking agents can also cause viscosity increases, which can change the flow characteristics of the test strip.
Moreover, in many of the prior art devices the labeled detection reagent is transported to the test zone at the solvent front. As a result, these prior art assay devices may exhibit uneven color banding or streaking due to the variable flow rate and characteristics at the solvent front, where the elution of the labeled reagent can be uneven. The streaking or color banding is undesirable as it can increase background, and can render reading a result difficult, due to uneven labeling in the test zone, and also due to the appearance of faintly positive weak test lines which can be difficult to interpret.
In some prior art test devices, particularly those used for ElAs and ELISAs, liquid carriers such as water, buffers and the like must be added to the test strip or to the sample during the test operation, which increases the possibility of operator error, and increases the complexity of the test operation. Moreover, elimination of reagents such as blocking agents, glazing agents, and facilitating agents during the manufacturing process may provide improved assay sensitivity, reproducibility and reliability, and overall a more efficient and economical assay production.